Blood coagulation is a complex system involving a large number of proteins that function in concert with platelets to yield hemostasis. The coagulation system is strictly regulated by a series of anticoagulant proteins present in plasma and on the surface of endothelial blood cells (Esmon, J. Biol. Chem. 264 (1989) 4743–4746; Bauer, Sem. Hematol. 28 (1991)10–18; and Rapaport, Blood 73 (1989) 35965). Under physiological conditions, pro- and anti-coagulant mechanisms are delicately balanced to provide hemostasis and coagulation. Disturbances in this balance result in either bleeding or thromboembolic disorders.
The present invention is related to a novel activity involved in a physiologically important anticoagulant system associated with Protein C and Protein S that has been elucidated in recent years and is shown as part of the blood coagulation interactions illustrated in FIG. 5.
In the above mentioned anticoagulant system, Protein C, a vitamin K-dependent plasma protein, is a key component that, after activation to Activated Protein C (APC) on endothelial cells by the thrombin/thrombomodulin complex, selectively degrades the coagulation Factors Va and VIIIa, i.e., the activated forms of the coagulation Factors V and VIII, respectively. (Esmon, supra; Stenflo, in Protein C and Related Proteins, ed. Bertina (Churchill Livingstone Longham Group, UK) (1988) 21–54; Mann et al., Ann. Rev. Biochem. 57 (1988) 915–956; and Kane et al., Blood 71 (1988) 539–55).
The activity of APC is influenced by another vitamin K-dependent plasma protein, designated Protein S, which functions as a cofactor to APC in the degradation of Factors Va and VIIIa (Esmon, supra; Stenflo, supra; and Dahlbäck, Thromb. Haemostas. 66 (1991) 49–61).
The above mentioned Factors Va and VIIIa are phospholipid-bound cofactors involved in the activation of Factor X and prothrombin, respectively, and are, thus, indirectly involved in the conversion of fibrinogen to fibrin, i.e., in clot formation. Accordingly, the rate of the coagulation reaction is dependent on the balance between the activation of Factors VIII and V and the degradation of their activated forms, the unactivated Factors VIII and V being poor substrates for APC.
Disturbances in the blood coagulation system are frequently manifested as serious and often life-threatening conditions, and knowledge about the underlying causes for the disturbances is often crucial in order to enable diagnosis and/or successful therapy of a manifested disease or the screening of individuals having a predisposition for a blood coagulation disease. For instance, therapeutic use of purified Protein C has been developed as a result of the discovery of Protein C deficiency associated with thrombophilia.
Thrombophilia can be defined as a tendency towards early-onset venous thomboembolic disease in adults in the absence of known risk factors. Although abnormalities have been determined for some thrombophilic patients, in the majority of such cases no laboratory test abnormalities were identified.
The present invention is related to a new defect in anticoagulant response to activated Protein C, called APC-resistance, which has been shown to be inherited and associated with familial thrombophilia.
In a few cases thrombophilia has been associated with hypothetical factors, such as an anti-Protein C antibody (Mitchell et al., New England Journal of Medicine, 1987, Vol. 316, 1638–1642), an anti-cardiolipin antibody (Amer et al., Thrombosis Research 57 (1990) 247–258) and a defective Factor VIII molecule (Dahlbäck et al., Thromb. Haemost. 65, Abstract 39, 658 (1991)).
In WO 93/10261, in vitro methods for the diagnosis of a manifested blood coagulation disorder or for the screening of individuals predisposed to a blood coagulation disorder are disclosed. These methods are based on measurement of the anticoagulant response to exogenous APC added to a plasma sample from the individual to be tested, a weak anticoagulant response to APC, i.e., APC-resistance, indicating manifestation of or predisposition to blood coagulation disorders, and especially a thromboembolic disease. No explanation for APC-resistance is given but the resistance to APC is suggested to be due to unknown interactions in the blood coagulation system or to unknown coagulation factor(s) thereof. However, several possible explanations connecting the APC-resistance to functional Protein S deficiency, a Protein C inhibitory antibody, a protease inhibitor for APC or a mutation giving an APC-resistant Factor Va molecule or a Factor VIII gene mutation were ruled out.